Anti‐collagen type II antibodies determination
Circulating anti‐collagen type II antibodies were determined by homemade enzyme‐linked immunosorbent assay (ELISA). ELISA plates were coated with collagen type II [10 µg/ml phosphate‐buffered saline (PBS)]. Following overnight incubation at 4°C, the plates were blocked with 3% bovine serum albumin (BSA). Mouse plasma at dilution of 1:800 were added to the blocked ELISA plates and incubated for 2 h at room temperature (n  = 10 for each treatment group). The binding was probed with goat anti‐mouse IgG conjugated with alkaline phosphatase (Jackson ImmunoResearch Laboratories, West Grove, PA, USA), followed by the addition of appropriate substrate. The data were read by ELISA reader at optical density (OD) 405 nm and presented as OD at 405nm.